Compositions comprising citrulline and leucine and their use in the treatment of diabetes and metabolic syndrome

ABSTRACT

Compositions contain citrulline and leucine or a metabolite thereof, such as hydroxyisocaproic acid (HICA) or beta-hydroxy-beta-methylbutyrate (HMB), including, for example, a synergistic amount of citrulline and leucine. Such compositions may be used in methods for treatment of pre-diabetes, metabolic syndrome, or diabetes.

PRIORITY CLAIM

The present application is a divisional of U.S. patent application Ser.No. 15/892,078 filed Feb. 8, 2018, which is a divisional of U.S. patentapplication Ser. No. 15/027,580 filed Apr. 6, 2016, now U.S. Pat. No.9,913,818 issued Mar. 13, 2018, which is a National Stage ofInternational Application No. PCT/EP2014/071230 filed Oct. 3, 2014,which claims priority to U.S. Provisional Patent App. No. 61/888,636filed Oct. 9, 2013, the entire contents of which are incorporated hereinby reference.

BACKGROUND

The present disclosure generally relates to methods and compositions fortreatment or prevention of pre-diabetes, diabetes, hyperinsulinemia,hyperglycemia, and metabolic syndrome. More specifically, the presentdisclosure relates to compositions that comprise a synergistic amount ofboth citrulline and leucine.

Insulin resistance is associated with a number of disease states andconditions and is present in approximately 30-40% of non-diabeticindividuals. These disease states and conditions include, but are notlimited to, pre-diabetes and metabolic syndrome (also referred to asinsulin resistance syndrome). Pre-diabetes is a state of abnormalglucose tolerance characterized by either impaired glucose tolerance(IGT) or impaired fasting glucose (IFG). Patients with pre-diabetes areinsulin resistant and are at high risk for future progression to overtType 2 diabetes. Metabolic syndrome is an associated cluster of traitsthat include, but is not limited to, hyperinsulinemia, abnormal glucosetolerance, obesity, redistribution of fat to the abdominal or upper bodycompartment, hypertension, dysfibrinolysis, and a dyslipidemiacharacterized by high triglycerides, low HDL-cholesterol, and smalldense LDL particles. Insulin resistance has been linked to each of thetraits, suggesting metabolic syndrome and insulin resistance areintimately related to one another.

SUMMARY

The present inventors surprisingly and unexpectedly found thatadministration of citrulline and leucine (or a metabolite thereof) has asynergistic effect and is able to reduce fasting glucose levels andfasting insulin plasma levels below the level of reduction obtained withindividual administration of citrulline or leucine.

The present disclosure relates to methods of treating or preventing adisease or condition in a subject in need thereof (e.g., an obese oroverweight subject) comprising administering to the subject atherapeutically effective amount of citrulline and a therapeuticallyeffective amount of leucine (or a metabolite thereof). Thetherapeutically effective amount of citrulline and a therapeuticallyeffective amount of leucine (or a metabolite thereof) may beadministered to the subject at the same time or at a different time andmay be administered in amounts that have a synergistic effect.

The present disclosure provides methods of treating or preventing adisease or condition in a subject in need thereof comprisingadministering to the subject a therapeutically effective amount ofcitrulline and a therapeutically effective amount of leucine (or ametabolite thereof).

In some embodiments, the disease or condition is selected from the groupconsisting of: pre-diabetes, diabetes, hyperinsulinemia, hyperglycemia,metabolic syndrome, obesity, and combinations thereof.

In an embodiment, the disease or condition is metabolic syndrome.

In another embodiment, the disease or condition is hyperinsulinemia.

In another embodiment, the disease or condition is hyperglycemia.

In another embodiment, the metabolite of leucine is hydroxy isocaproicacid (HICA) or beta-hydroxy-beta-methylbutyrate (HMB).

In another embodiment, the therapeutically effective amount ofcitrulline and the therapeutically effective amount of leucine (or ametabolite thereof) are present in a synergistic amount.

In another embodiment, citrulline and leucine (or a metabolite thereof)is administered to the subject weekly, daily, two times a day, or threetimes a day.

In another embodiment, the subject is administered a fixed amountcitrulline and leucine (or a metabolite thereof).

In another embodiment, the subject is administered a weight-based doseof citrulline and leucine (or a metabolite thereof).

In another embodiment, the weight-based dose is about 0.01 g/kg to about0.3 g/kg.

In another embodiment, a pharmaceutical composition is administered tothe subject that comprises both the therapeutically effective amount ofcitrulline and the therapeutically effective amount of leucine (or ametabolite thereof).

In another embodiment, the therapeutically effective amount ofcitrulline and the therapeutically effective amount of leucine (or ametabolite thereof) are administered orally.

In another embodiment, the method is effective to achieve at least oneof the following modifications: reduction in fasting blood sugar level,decrease in insulin resistance, reduction of hypoinsulinemia,improvement in glucose tolerance, or any combination thereof.

In another embodiment, the therapeutically effective amount ofcitrulline and the therapeutically effective amount of leucine (or ametabolite thereof) is administered in conjunction with at least oneadditional treatment method, said additional treatment method comprisingadministering at least one pharmaceutical composition comprising anactive agent selected from the group consisting of: a sulfonylurea, ameglitinide, a biguanide, an alpha-glucosidase inhibitor, athiazolidinedione, a DPP-IV inhibitor, a glucagon-like peptide (GLP)-1analog, or insulin.

The present disclosure also provides a composition, nutritionalcomposition, or medical food comprising citrulline and leucine (or ametabolite thereof) useful for the treatment of pre-diabetes, diabetes,hyperinsulinemia, hyperglycemia, metabolic syndrome, obesity, or anycombination thereof.

In an embodiment, the metabolite of leucine is hydroxy isocaproic acid(HICA) or beta-hydroxy-beta-methylbutyrate (HMB).

In another embodiment, citrulline and leucine are present in asynergistic amount.

In another embodiment, citrulline and leucine are both present in anamount effective to treat metabolic syndrome, hyperglycemia, orhyperinsulinemia.

In another embodiment, the composition is a pharmaceutical composition.

These and other embodiments of the disclosure are described in furtherdetail herein below.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing summary, as well as the following detailed description ofthe disclosure, will be better understood when read in conjunction withthe appended figures. For the purpose of illustrating the disclosure,shown in the figures are embodiments which are presently preferred. Itshould be understood, however, that the disclosure is not limited to theprecise arrangements, examples and instrumentalities shown.

FIG. 1 shows fasting glucose levels in rats fed a high fat diet (HFD)and treated with 0.2 or 1.0 gkg/day of citrulline, 1.0 g/kg/day ofleucine or 1.0 g/kg/day of both citrulline and leucine. P<0.05 versusHFD.

FIG. 2 shows fasting insulin plasma levels in rats fed a high fat diet(HFD) and treated with 0.2 or 1.0 gkg/day of citrulline, 1.0 g/kg/day ofleucine or 1.0 g/kg/day of both citrulline and leucine. P<0.05 versusHFD.

DETAILED DESCRIPTION

All dosage ranges contained within this application are intended toinclude all numbers, whole or fractions, contained within said range.All percentages expressed herein are by weight of the total weight ofthe fiber blend unless expressed otherwise. As used herein, “about” isunderstood to refer to numbers in a range of numerals. Moreover, allnumerical ranges herein should be understood to include all integer,whole or fractions, within the range.

As used herein, “subject” includes, but is not limited to, mammals,which include but is not limited to, rodents, aquatic mammals, domesticanimals such as dogs and cats, farm animals such as sheep, pigs, cowsand horses, and humans. A subject may include a “patient” which isunderstood to include an animal, especially a mammal, and moreespecially a human that is receiving or intended to receive treatment,as treatment is herein defined. While the terms “individual” and“patient” are often used herein to refer to a human, the presentdisclosure is not so limited. Accordingly, the terms “individual” and“patient” refer to any animal, mammal or human, having or at risk for amedical condition that can benefit from the treatment.

“Nutritional compositions,” as used herein, are understood to includeany number of optional additional ingredients, including conventionalfood additives, for example one or more, acidulants, additionalthickeners, buffers or agents for pH adjustment, chelating agents,colorants, emulsifies, excipient, flavor agent, mineral, osmotic agents,a pharmaceutically acceptable carrier, preservatives, stabilizers,sugar, sweeteners, texturizers, and/or vitamins. The optionalingredients can be added in any suitable amount.

The terms “prevention”, “prevent”, “preventing”, “suppression”,“suppress”, “suppressing”, “inhibit” and “inhibition” as used hereinrefer to a course of action (such as administering a compound orpharmaceutical composition) initiated in a manner (e.g., prior to theonset of a clinical symptom of a disease state or condition) so as toprevent, suppress or reduce, either temporarily or permanently, theonset of a clinical manifestation of the disease state or condition.Such preventing, suppressing or reducing need not be absolute to beuseful.

The terms “treatment”, “treat” and “treating” as used herein refers acourse of action (such as administering a compound or pharmaceuticalcomposition) initiated after the onset of a clinical symptom of adisease state or condition so as to eliminate, reduce, suppress orameliorate, either temporarily or permanently, a clinical manifestationor progression of the disease state or condition. Such treating need notbe absolute to be useful.

As used herein, the term “in need of treatment” refers to a judgmentmade by a caregiver that a patient requires or will benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that includes the knowledgethat the patient is ill, or will be ill, as the result of a conditionthat is treatable by a method or compound of the disclosure.

As used herein, the term “in need of prevention” refers to a judgmentmade by a caregiver that a patient requires or will benefit fromprevention. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that includes the knowledgethat the patient will be ill or may become ill, as the result of acondition that is preventable by a method or compound of the disclosure.

As used herein, the term “therapeutically effective amount” refers to anamount of citrulline or leucine (or a metabolite thereof), either aloneor as a part of a pharmaceutical composition, that is capable of havingany detectable, positive effect on any symptom, aspect, orcharacteristics of a disease state or condition when administered to apatient (e.g., as one or more doses). Such effect need not be absoluteto be beneficial.

The term “insulin resistance” as used herein refers to a condition wherea normal amount of insulin is unable to produce a normal physiologicalor molecular response. In some cases, a hyper-physiological amount ofinsulin, either endogenously produced or exogenously added, is able toovercome the insulin resistance in whole or in part and produce abiologic response.

Methods of treating or preventing a disease or condition (e.g.,hyperinsulinemia, hyperglycemia, metabolic syndrome, Type 2 diabetes,Type 1 diabetes, and obesity) in a subject in need thereof, are providedby the disclosure and comprise administering to the subject atherapeutically effective amount of citrulline and a therapeuticallyeffective amount of leucine (or a metabolite thereof) (e.g. hydroxyisocaproic acid (HICA) or beta-hydroxy-beta-methylbutyrate (HMB)). Theinventors have unexpectedly found that citrulline and leucine (or ametabolite thereof) when administered to a subject reduces fastingplasma glucose levels and fasting plasma insulin levels below thatachieved with administration of either citrulline or leucine alone(i.e., exhibits a synergistic effect). In some embodiments, the methodis sufficient to achieve at least one of the following modifications:reduction in fasting blood sugar level, decrease in insulin resistance,reduction of hyperinsulinemia, and/or improvement in glucose tolerance.In some embodiments, the treatment or prevention method reduces asymptom of diabetes or the chances of developing a symptom of diabetes,wherein the symptom is selected from the group consisting ofatherosclerosis, obesity, hypertension, hyperlipidemia, fatty liverdisease, nephropathy, neuropathy, retinopathy, foot ulceration andcataracts, associated with diabetes. Optionally, administration of thetherapeutically effective amount of citrulline and the therapeuticallyeffective amount of leucine (or a metabolite thereof) results in animprovement in glycemic control in the subject.

The present disclosure also provides compositions (e.g., pharmaceuticalcompositions) that comprise citrulline and leucine (or a metabolitethereof) (e.g., hydroxy isocaproic acid (HICA) orbeta-hydroxy-beta-methylbutyrate (HMB)) including, where citrulline andleucine (or a metabolite thereof) are present in a synergistic amount.

The present disclosure also provides nutritional supplements and medicalfoods that comprise citrulline and leucine (or a metabolite thereof)(e.g., hydroxy isocaproic acid (HICA) orbeta-hydroxy-beta-methylbutyrate (HMB)) including, where citrulline andleucine (or a metabolite thereof) are present in a synergistic amount.

Citrulline and leucine (or a metabolite thereof) for use according tothe present disclosure can be formulated in compositions, especiallypharmaceutical compositions, for use in the methods herein. Suchcompositions comprise a therapeutically or prophylactically effectiveamount (e.g., a synergistic amount) of citrulline and leucine (or ametabolite thereof) in admixture with a suitable carrier, e.g., apharmaceutically acceptable agent.

Pharmaceutically acceptable agents include carriers, excipients,diluents, antioxidants, preservatives, coloring, flavoring and dilutingagents, emulsifying agents, suspending agents, solvents, fillers,bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents,wetting agents, complexing agents, buffering agents, antimicrobials, andsurfactants.

Neutral buffered saline or saline mixed with albumin are exemplaryappropriate carriers. The pharmaceutical compositions can includeantioxidants such as ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, pluronics, orpolyethylene glycol (PEG). Also by way of example, suitable tonicityenhancing agents include alkali metal halides (preferably sodium orpotassium chloride), mannitol, sorbitol, and the like. Suitablepreservatives include benzalkonium chloride, thimerosal, phenethylalcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid andthe like. Hydrogen peroxide also can be used as preservative. Suitablecosolvents include glycerin, propylene glycol, and PEG. Suitablecomplexing agents include caffeine, polyvinylpyrrolidone,beta-cyclodextrin or hydroxy-propyl-beta-cyclodextrin. Suitablesurfactants or wetting agents include sorbitan esters, polysorbates suchas polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, andthe like. The buffers can be conventional buffers such as acetate,borate, citrate, phosphate, bicarbonate, or Tris-HCl. Acetate buffer maybe about pH 4-5.5, and Tris buffer can be about pH 7-8.5. Additionalpharmaceutical agents are set forth in Remington's PharmaceuticalSciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company,1990.

The composition can be in liquid form or in a lyophilized orfreeze-dried form and may include one or more lyoprotectants,excipients, surfactants, high molecular weight structural additivesand/or bulking agents (see for example U.S. Pat. Nos. 6,685,940,6,566,329, and 6,372,716). In one embodiment, a lyoprotectant isincluded, which is a non-reducing sugar such as sucrose, lactose ortrehalose. The amount of lyoprotectant generally included is such that,upon reconstitution, the resulting formulation will be isotonic,although hypertonic or slightly hypotonic formulations also may besuitable. Exemplary lyoprotectant concentrations for sugars (e.g.,sucrose, lactose, trehalose) in the pre-lyophilized formulation are fromabout 10 mM to about 400 mM. In another embodiment, a surfactant isincluded, such as for example, nonionic surfactants and ionicsurfactants such as polysorbates (e.g. polysorbate 20, polysorbate 80);poloxamers (e.g. poloxamer 188); poly(ethylene glycol) phenyl ethers(e.g. Triton); sodium dodecyl sulfate (SDS); sodium laurel sulfate;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the MONAQUAT® series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc). Exemplaryamounts of surfactant that may be present in the pre-lyophilizedformulation are from about 0.001-0.5%. High molecular weight structuraladditives (e.g. fillers, binders) may include for example, acacia,albumin, alginic acid, calcium phosphate (dibasic), cellulose,carboxymethylcellulose, carboxymethylcellulose sodium,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, microcrystalline cellulose, dextran,dextrin, dextrates, sucrose, tylose, pregelatinized starch, calciumsulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose,disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite,polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose,compressible sugar, magnesium aluminum silicate, maltodextrin,polyethylene oxide, polymethacrylates, povidone, sodium alginate,tragacanth microcrystalline cellulose, starch, and zein. Exemplaryconcentrations of high molecular weight structural additives are from0.1% to 10% by weight. In other embodiments, a bulking agent (e.g.,mannitol, glycine) may be included.

Compositions are suitable for administration to a subject by any routeavailable to the skilled worker, such as intraarticular, subcutaneous,intravenous, intramuscular, intraperitoneal, intracerebral(intraparenchymal), intracerebroventricular, intramuscular, intraocular,intraarterial, intralesional, intrarectal, transdermal, oral, andinhaled routes. A parenteral formulation typically will be a sterile,pyrogen-free, isotonic aqueous solution, optionally containingpharmaceutically acceptable preservatives.

Suitable and/or preferred pharmaceutical formulations can be determinedin view of the present disclosure and general knowledge of formulationtechnology, depending upon the intended route of administration,delivery format, and desired dosage. Regardless of the manner ofadministration, an effective dose can be calculated according to patientbody weight, body surface area, or organ size. Further refinement of thecalculations for determining the appropriate dosage for treatmentinvolving each of the formulations described herein are routinely madein the art and is within the ambit of tasks routinely performed in theart. Appropriate dosages can be ascertained through use of appropriatedose-response data.

The pharmaceutical compositions can comprise citrulline and leucine (ora metabolite thereof) in combination with other active agents. Suchcombinations are those useful for their intended purpose. Thecombinations which are part of this disclosure can be citrulline andleucine (or a metabolite thereof) and at least one additional agentselected from the lists below. The active agents set forth below areillustrative for purposes and not intended to be limited. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

The disclosure further contemplates that pharmaceutical compositionscomprising one or more other active agents may be administeredseparately from citrulline and leucine (or a metabolite thereof), andsuch separate administrations may be performed at the same point ordifferent points in time, such as for example the same or differentdays.

Alternatively or in addition, therapeutic treatment with at least one ormore additional active agents may be used which may act via differentmodes of action: 1) sulfonylureas (e.g., chlorpropamide, tolazamide,acetohexamide, tolbutamide, glyburide, glimepiride, glipizide) and/ormeglitinides (e.g., repaglinide, nateglinide) that essentially stimulateinsulin secretion; 2) biguanides (e.g., metformin) act by promotingglucose utilization, reducing hepatic glucose production and diminishingintestinal glucose output; 3) alpha-glucosidase inhibitors (e.g.,acarbose, miglitol) slow down carbohydrate digestion and consequentlyabsorption from the gut and reduce postprandial hyperglycemia; 4)thiazolidinediones (e.g., troglitazone, pioglitazone, rosiglitazone,glipizide, balaglitazone, rivoglitazone, netoglitazone, troglitazone,englitazone, AD 5075, T 174, YM 268, R 102380, NC 2100, NIP 223, NIP221, MK 0767, ciglitazone, adaglitazone, CLX 0921, darglitazone, CP92768, BM 152054) that enhance insulin action, thus promoting glucoseutilization in peripheral tissues; 5) glucagon-like-peptides includingDPP4 inhibitors (e.g., sitagliptin); and 6) insulin, which stimulatestissue glucose utilization and inhibits hepatic glucose output.Glucagon-like peptide-1 (GLP-1) and analogs, DPP-IV-resistant analogues(incretin mimetics), DPP-IV inhibitors, insulin, insulin analogues, PPARgamma agonists, dual-acting PPAR agonists, GLP-1 agonists or analogues,PTP1B inhibitors, SGLT inhibitors, insulin secretagogues, RXR agonists,glycogen synthase kinase-3 inhibitors, insulin sensitizers, immunemodulators, beta-3 adrenergic receptor agonists, Pan-PPAR agonists, 1lbeta-HSD1 inhibitors, amylin analogues, biguanides, alpha-glucosidaseinhibitors, meglitinides, thiazolidinediones, sulfonylureas and the likealso may be used as the other active agent(s) (see for example Nathan,2006, N. Engl. J. Med. 355:2477-2480; Kahn et al., 2006, N. Engl. J.Med. 355:2427-2443). In yet another embodiment, the active agent may bean HMG Co-A reductase inhibitor (e.g., statins).

The pharmaceutical compositions used in the disclosure may include atherapeutically effective amount or a prophylactically effective amountof citrulline and leucine (or a metabolite thereof). A therapeuticallyeffective amount refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects are outweighed by the therapeutically beneficialeffects. A prophylactically effective amount refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result.

The present disclosure also provides a nutritional composition or amedical food comprising citrulline and leucine (or a metabolitethereof). Such compositions and foods comprise a therapeutically orprophylactically effective amount (e.g., a synergistic amount) ofcitrulline and leucine (or a metabolite thereof). In an embodiment, thenutritional composition comprises at least about 0.01 g to about 0.3 gof citrulline and at least about 0.01 g to about 0.3 g of leucine (or ametabolite thereof).

The nutritional composition can contain a protein source. Any type ofdietary protein may be used, provided that the minimum requirements foressential amino acid content are met. However, in an embodiment, morethan 50% or more than 60% by weight of the protein source is whey (henceensuring a best balanced amino-acid profile). Thus, protein sourcesbased on whey, casein and mixtures thereof may be used as well asprotein sources based on soy, pea, rice or potato (and not restricted tothese ones). Regarding whey proteins, the protein source may be based onacid whey or sweet whey or mixtures thereof and may includealpha-lactalbumin and beta-lactoglobulin in whatever proportions aredesired.

The nutritional composition can contain a carbohydrate source. Anycarbohydrate source conventionally found in nutritional compositionssuch as lactose, saccharose, maltodextrin, starch and mixtures thereof,may be used, although the preferred source of carbohydrates is lactose.

In an embodiment, the nutritional composition further comprises fats,such as, for example, fish oils or nonmarine oils or medium chaintriglyceride (MCT). Non-limiting examples of fish oils includedocosahexaenoic acid (“DHA”) and eicosapentaenoic acid (“EPA”).Alternatively or additionally, DHA and EPA may be present from anon-fish oil source, e.g., algae, modified plants, and the like.

In some embodiments, the nutritional composition further comprisesantioxidants. Non-limiting examples of suitable antioxidants includesubstances that inhibit oxidation or reactions promoted by ReactiveOxygen Species (“ROS”) and other radical and non-radical species andalso include molecules capable of slowing or preventing the oxidation ofother molecules. For example, in addition to the fiber blend, thenutritional composition can further comprise carotenoids, coenzyme Q10(“CoQ10”), flavonoids, glutathione Goji (wolfberry), hesperidine,lactowolfberry, lignin, lutein, lycopene, polyphenols, selenium, vitaminA, vitamin B₁, vitamin B₆, vitamin B₁₂, vitamin C, vitamin D, vitamin E,zeaxanthin, or combinations thereof.

In some embodiments, the nutritional composition further comprisesvitamins, minerals or combinations thereof. Vitamins include fat-solubleor water-soluble organic substances essential in minute amounts fornormal growth and activity of the body. The vitamins used in thecomposition are obtained naturally from plant and animal foods orsynthetically made and can include pro-vitamins, derivatives andanalogs. Non-limiting examples of suitable vitamins include Vitamin A,Vitamin B₁ (thiamine), Vitamin B₂ (riboflavin), Vitamin B₃ (niacin orniacinamide), Vitamin B₅ (pantothenic acid), Vitamin B₆ (pyridoxine,pyridoxal, or pyridoxamine, or pyridoxine hydrochloride), Vitamin B₇(biotin), Vitamin B₉ (folic acid), and Vitamin B₁₂ (various cobalamins;commonly cyanocobalamin in vitamin supplements), Vitamin C, Vitamin D,Vitamin E, Vitamin K, folic acid, biotin and combinations thereof.Non-limiting examples of suitable minerals include boron, calcium,chromium, copper, iodine, iron, magnesium, manganese, molybdenum,nickel, phosphorus, potassium, selenium, silicon, tin, vanadium, zincand combinations thereof.

In an embodiment, the nutritional composition further comprisesphytonutrients that are health-promoting compounds from plant sources.The phytonutrients that can be used in the nutritional compositions areany chemicals produced by a plant that impart one or more healthbenefits on the user. Non-limiting examples of suitable phytonutrientsinclude phenolic compounds, terpenes, betalains, organosulfides, proteininhibitors, other organic acids and combinations thereof.

The nutritional composition may contain emulsifiers and stabilizers suchas soy lecithin, citric acid esters of mono- and di-glycerides, and thelike. Furthermore, the nutritional composition may optionally containother substances which may have a beneficial effect such as lactoferrin,nucleotides, nucleosides, and the like.

In an embodiment, the nutritional compositions comprise a prebiotic inaddition to the 2-fucosylated oligosaccharide. A prebiotic is a foodsubstance that selectively promotes the growth of beneficial bacteria orinhibits the growth or mucosal adhesion of pathogenic bacteria in theintestines. They are not inactivated in the stomach and/or upperintestine or absorbed in the gastrointestinal tract of the personingesting them, but they are fermented by the gastrointestinalmicroflora and/or by probiotics. Prebiotics are, for example, defined byGlenn Gibson et al., “Dietary Modulation of the Human ColonicMicrobiota: Introducing the Concept of Prebiotics,” J. Nutr., 125:1401-1412 (1995).

Non-limiting examples of prebiotics include acacia gum, alpha glucan,arabinogalactans, beta glucan, dextrans, fructooligosaccharides (FOS),galactooligosaccharides (GOS), fucosyllactoses, galactomannans,gentiooligosaccharides, glucooligosaccharides, guar gum, inulin,isomaltooligosaccharides, lactoneotetraose, lactosucrose, lactulose,levan, maltodextrins, milk oligosaccharides, partially hydrolyzed guargum, pecticoligosaccharides, resistant starches, retrograded starch,sialooligosaccharides, sialyllactose, soyoligosaccharides, sugaralcohols, xylooligosaccharides, or their hydrolysates, or combinationsthereof. The milk oligosaccharides may be bovine's milk oligosaccharides(BMOs) and/or human milk oligosaccharides (HMOs).

The nutritional composition may be in the form of tablets, capsules,pastilles or a liquid for example. The supplement may further containprotective hydrocolloids (such as gums, proteins, modified starches),binders, film forming agents, encapsulating agents/materials, wall/shellmaterials, matrix compounds, coatings, emulsifiers, surface activeagents, solubilizing agents (oils, fats, waxes, lecithins or the like),adsorbents, carriers, fillers, co-compounds, dispersing agents, wettingagents, processing aids (solvents), flowing agents, taste maskingagents, weighting agents, jellifying agents and gel forming agents. Thesupplement may also contain conventional pharmaceutical additives andadjuvants, excipients and diluents, including, but not limited to,water, gelatin of any origin, vegetable gums, ligninsulfonate, talc,sugars, starch, gum arabic, vegetable oils, polyalkylene glycols,flavoring agents, preservatives, stabilizers, emulsifying agents,buffers, lubricants, colorants, wetting agents, fillers, and the like.

The nutritional composition can be added in a product acceptable to theconsumer (who is a human or an animal), such as an ingestible carrier orsupport, respectively. Examples of such carriers or supports are apharmaceutical or a food or a pet food composition. Non-limitingexamples for such compositions are milk, yogurt, curd, cheese, fermentedmilks, milk based fermented products, fermented cereal based products,milk based powders, human milk, preterm formula, infant formula, oralsupplement, and tube feeding.

Further, the nutritional composition may contain an organic or inorganiccarrier material suitable for oral or enteral administration as well asvitamins, minerals trace elements and other micronutrients in accordancewith the recommendations of government bodies such as the USRDA.

The nutritional composition may be prepared in liquid form. Water is themost common carrier for the components of the composition, but thecomposition can be provided in other liquids such as, for example, milk,fruit juice, coffee, tca or other beverages, when such compositions areorally administered. Water is typically used for enteral formulations.

The nutritional composition can be a dry powdered formulation. Thepowdered formulation can be made by combining dry powdered ingredientsor can be made from a liquid nutritional composition by drying theliquid composition with one of the processes known in the art, includingspray drying, freeze drying or other drying techniques, to produce a drypowdered composition. If desired, other nutritional components orcompositions can be added to the liquid prior to drying to provideenhanced nutritional benefits to the powdered formulation. Such powderedformulations have a much greater shelf life and can be packaged forstorage and transport for future use. At that time, the powderedformulations can be reconstituted with water or other fluids and thenadministered to the individual orally or enterally. The powderedformulation can be packaged in various containers, including those forbulk provision of such powdered formulations for adding to a liquid in aglass, bottle or other fluid containing vessel, or a single serving canbe provided with the powder present in a container to which water orother fluids can be added to form the liquid for oral or enteraladministration.

In an embodiment, the nutritional composition is a complete nutritionproduct that contains sufficient types and levels of macronutrients(protein, fats and carbohydrates) and micronutrients to be sufficient asa sole source of nutrition for the animal to which the composition isadministered. In another embodiment, the nutritional composition is anincomplete nutrition product that does not contain sufficient levels ofmacronutrients (protein, fats and carbohydrates) or micronutrients to besufficient as a sole source of nutrition for the animal to which thecomposition is administered.

The present disclosure also provides methods for treating or preventinga condition selected from the group consisting of metabolic syndrome,obesity, insulin resistance syndrome, diabetes (both primary essentialdiabetes such as Type II Diabetes and secondary nonessential diabetes)in a subject in need or treatment or in a subject in need of preventionby administering to the subject a synergistic amount of citrulline andleucine (or a metabolite thereof) (e.g., a composition comprising asynergistic amount of citrulline and leucine (or a metabolite thereof),or a nutritional supplement or medical food comprising a synergisticamount of citrulline and leucine (or a metabolite thereof)) effective totreat the condition. The methods of the disclosure may be effective torelieve a symptom of diabetes or the chance of developing a symptom ofdiabetes, such as atherosclerosis, hypertension, hyperlipidemia, fattyliver disease, nephropathy, neuropathy, retinopathy, foot ulceration andcataracts, each such symptom being associated with diabetes, can bereduced.

The synergistic amount of citrulline and leucine (or a metabolitethereof) can be administered by any conventional route of systemicadministration. Preferably they are administered orally. Other routes ofadministration that can be used in accordance with this disclosureinclude rectally, parenterally, by injection (e.g. intravenous,subcutaneous, intramuscular or intraperitioneal injection), or nasally.

In an embodiment, the synergistic amount of citrulline and leucine (or ametabolite thereof) is administered to a subject with at least one othermedically accepted treatment (e.g., medication, drug, therapeutic,active agent) for the disease, condition or complication. In anotherembodiment, the at least one other medically accepted treatment for thedisease, condition or complication is reduced or discontinued (e.g.,when the subject is stable), while treatment with the synergistic amountof citrulline and leucine (or a metabolite thereof) is maintained at aconstant dosing regimen. In another embodiment, the at least one othermedically accepted treatment for the disease, condition or complicationis reduced or discontinued (e.g., when the subject is stable), andtreatment with the synergistic amount of citrulline and leucine (or ametabolite thereof) is reduced (e.g., lower dose, less frequent dosing,shorter treatment regimen). In another embodiment, the at least oneother medically accepted treatment for the disease, condition orcomplication is reduced or discontinued (e.g., when the subject isstable), and treatment with the synergistic amount of citrulline andleucine (or a metabolite thereof) is increased (e.g., higher dose, morefrequent dosing, longer treatment regimen). In yet another embodiment,the at least one other medically accepted treatment for the disease,condition or complication is maintained and treatment with thesynergistic amount of citrulline and leucine (or a metabolite thereof)is reduced or discontinued (e.g., lower dose, less frequent dosing,shorter treatment regimen). In yet another embodiment, the at least oneother medically accepted treatment for the disease, condition orcomplication and treatment with the synergistic amount of citrulline andleucine (or a metabolite thereof) are reduced or discontinued (e.g.,lower dose, less frequent dosing, shorter treatment regimen). The oneother medically accepted treatment may include administering at leastone pharmaceutical composition comprising an active agent selected fromthe group consisting of: a sulfonylurea, a meglitinide, a biguanide, analpha-glucosidase inhibitor, a thiazolidinedione, a DPP-IV inhibitor, aglucagon-like peptide (GLP)-1 analog, insulin, or a combination thereof.

The disclosure further contemplates that the synergistic amount ofcitrulline and leucine (or a metabolite thereof) used in accordance withthe methods provided herein, may be administered in conjunction withmore traditional treatment methods and pharmaceutical compositions(e.g., active agents). Such compositions, may include for example,DPP-IV inhibitors, insulin, insulin analogues, PPAR gamma agonists,dual-acting PPAR agonists, GLP-1 agonists or analogues, PTP1Binhibitors, SGLT inhibitors, insulin secretagogues, RXR agonists,glycogen synthase kinase-3 inhibitors, insulin sensitizers, immunemodulators, beta-3 adrenergic receptor agonists, Pan-PPAR agonists, 1lbeta-HSDI inhibitors, amylin analogues, biguanides, alpha-glucosidaseinhibitors, meglitinides, thiazolidinediones, sulfonylureas and the like(see, e.g., Nathan, 2006, N. Engl. J. Med. 355:2477-2480; Kahn et al.,2006, N. Engl. J. Med. 355:2427-2443). In certain embodiments, thesynergistic amount of citrulline and leucine (or a metabolite thereof)used in accordance with the disclosure may prevent or delay the need foradditional treatment methods or pharmaceutical compositions. In otherembodiments, the synergistic amount of citrulline and leucine (or ametabolite thereof) may reduce the amount, frequency or duration ofadditional treatment methods or pharmaceutical compositions.

Methods of treating or preventing a disease or condition in accordancewith the present disclosure may use a pre-determined or “routine”schedule for administration of the synergistic amount of citrulline andleucine (or a metabolite thereof). As used herein a routine schedulerefers to a predetermined designated period of time between doseadministrations. The routine schedule may encompass periods of timewhich are identical or which differ in length, as long as the scheduleis predetermined. Any particular combination would be covered by theroutine schedule as long as it is determined ahead of time that theappropriate schedule involves administration on a certain day.

Alternatively, methods of treating or preventing a disease or conditionin accordance with the present disclosure may use a schedule foradministration of the synergistic amount of citrulline and leucine (or ametabolite thereof) that is based upon the presence of disease symptomsas a means to determine when to administer one or more subsequent doses.Similar, this approach may be used as a means to determine whether asubsequent dose should be increased or decreased, based upon the effectof a previous dose.

The compositions disclosed herein may be effective to improve glycemiccontrol. Assessment of improvement in glycemic control may be assessed,for example, based on a change in hemoglobin Alc (HbAlc, see for exampleReynolds et al., BMJ, 333(7568):586-589, 2006). Improvements (e.g.,decrease) in HbAlc that are indicative of therapeutic efficacy may varydepending on the initial baseline measurement in a patient, with alarger decrease often corresponding to a higher initial baseline and asmaller decrease often corresponding to a lower initial baseline. In oneaspect of the disclosure, the method should result in an HbAlc decreaseof at least about 0.5% (e.g., at least about 0.5%, at least about 1%, atleast about 1.5%, at least about 2%, at least about 2.5%, at least about3%, at least about 3.5%, at least about 4% or more) compared withpre-dose levels.

One or more of the following secondary endpoints also may be determinedin order to assess efficacy of the treatment, such as for examplefasting blood sugar (e.g., glucose) levels (e.g., decrease to <130,<125, <120, <115, <110, <105, <100; alternatively decreaseof >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95% compared topre-dose levels), 120 minute oral glucose tolerance test (OGTT) (e.g.,<200, <190, <180, <170, <160, <150, <140), glucose/insulin C-peptide AUC(e.g., >25%, >50%, >60%, >70%, >80%, >90%, >100% increase frompre-treatment), reduction in diabetes medication (e.g., insulin, oralhypoglycemic agent), improvement in insulin sensitivity,

Similarly, assessment of efficacy for other diseases or conditions mayuse one or more of the aforementioned endpoints and/or others known inthe art. For example, the effect on hyperglycemia can be assessed bymeasuring fasting blood sugar (i.e., glucose) levels, the effect onhyperinsulinemia may be assessed by measuring insulin levels and/orC-peptide levels, and the effect on insulin resistance may be assessedby OGTT.

Similarly, subjects treated in accordance with the present disclosuremay experience a decrease in insulin resistance. Such decrease ininsulin resistance may be measured by an improvement in a homeostasismodel assessment (HOMA), an insulin tolerance test, an insulinsuppression test, a steady-state plasma glucose method, or any of theother assay methods know in the art (see, e.g., Matthews et al., 1985,Diabetologia 28:412-419; Odegaard et al., 2007, Nature 447:1116-1121;Emoto et al., 1999, Diabetes Care 22:818-822). Other of theaforementioned measurements may be made using any of a variety ofstandard assays known in the art, for example assays published inChemecky C C, Berger B J, eds. (2004). Laboratory Tests and DiagnosticProcedures, 4th ed. Philadelphia: Saunders; Fischbach F T, Dunning M BIII, eds. (2004). Manual of Laboratory and Diagnostic Tests, 7th ed.Philadelphia: Lippincott Williams and Wilkins; Genest J, et al. (2003).Recommendations for the management of dyslipidemia and the prevention ofcardiovascular disease: Summary of the 2003 update. Canadian MedicalAssociation Journal, 169(9): 921-924. Handbook of Diagnostic Tests(2003). 3rd ed. Philadelphia: Lippincott Williams and Wilkins; andPagana K D, Pagana T J (2002). Mosby's Manual of Diagnostic andLaboratory Tests, 2nd ed. St. Louis: Mosby.

The present disclosure also provides methods for selling a productcontaining a synergistic amount of citrulline and leucine (or ametabolite thereof) comprising the step of promoting that thesynergistic amount of citrulline and leucine (or a metabolite thereof)improves reduces fasting plasma glucose levels, reduces fasting plasmainsulin levels, and/or improves glycemic control in a human.

Promotion of the composition comprising citrulline and leucine (or ametabolite thereof) including, a synergistic amount of citrulline andleucine (or a metabolite thereof), may be made by advertising campaigns.These campaigns may consist of print, television, radio, and/or internetadvertising. Additionally or alternatively, promotion of the compositioncomprising citrulline and leucine (or a metabolite thereof) may includeplacing advertisements about the composition comprising citrulline andleucine (or a metabolite thereof) in journals and/or direct sale callsto consumers. Such promotional efforts may be directed to health careproviders, including physicians, nurses and/or hospitals. Additionallyor alternatively, promotional efforts may be directed to patients,including patients with a disease or disorder that may be treated withthe composition comprising citrulline and leucine (or a metabolitethereof). The step of promoting the composition comprising citrullineand leucine (or a metabolite thereof) may include promoting the benefitsof a product including a composition comprising citrulline and leucine(or a metabolite thereof) comprising the step of stating that thecomposition comprising citrulline and leucine (or a metabolite thereof)can be used to treat a metabolic disease, hyperinsulinemia, orhyperglycemia.

Without further description, it is believed that one of ordinary skillin the art may, using the preceding description and the followingillustrative examples, make and utilize the agents of the presentdisclosure and practice the claimed methods. The following workingexamples are provided to facilitate the practice of the presentdisclosure, and are not to be construed as limiting in any way theremainder of the disclosure

EXAMPLES Example 1: Administration of a Composition ComprisingCitrulline and Leucine Reduces Fasting Glucose and Insulin Plasma Levels

Compositions comprising citrulline, leucine, or citrulline or leucinewere tested for their effect on fasting glucose levels and insulinplasma levels. Briefly, rats were split into six groups and fed eitherchow, a high fat diet, a high fat diet with 1 g/kg leucine daily, a highfat diet with 0.2 g/kg citrulline, a high fat diet with 1 g/kgcitrulline daily or a high fat diet with a composition comprising 1.0g/kg leucine and 1.0 g/kg citrulline. Rats fed a high fat diet andeither 1 g/kg of citrulline or 1 g/kg leucine exhibited a decrease infasting plasma glucose levels and insulin plasma levels as compared torats fed a high fat diet. Only the administration of a compositioncomprising both citrulline and leucine achieved a significant result onglycemia. For plasma insulin level, citrulline alone or leucine alonewere efficient but unexpectedly only the co administration of bothleucine and citrulline allowed to completely normalised insulinemia atthe level observed in healthy rats eating a standard chow diet, thussuggesting a synergic effect between citrulline and leucine (FIG. 1 andFIG. 2).

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the disclosure and does not pose alimitation on the scope of the disclosure otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the disclosure.

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group can be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the disclosureto be practiced otherwise than specifically described herein.Accordingly, this disclosure includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by thedisclosure unless otherwise indicated herein or otherwise clearlycontradicted by context.

Specific embodiments disclosed herein can be further limited in theclaims using consisting of or and consisting essentially of language.When used in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharactcristic(s). Embodiments of the disclosure so claimed arcinherently or expressly described and enabled herein.

It is to be understood that the embodiments of the disclosure disclosedherein are illustrative of the principles of the present disclosure.Other modifications that can be employed are within the scope of thedisclosure. Thus, by way of example, but not of limitation, alternativeconfigurations of the present disclosure can be utilized in accordancewith the teachings herein. Accordingly, the present disclosure is notlimited to that precisely as shown and described.

While the present disclosure has been described and illustrated hereinby references to various specific materials, procedures and examples, itis understood that the disclosure is not restricted to the particularcombinations of materials and procedures selected for that purpose.Numerous variations of such details can be implied as will beappreciated by those skilled in the art. It is intended that thespecification and examples be considered as exemplary, only, with thetrue scope and spirit of the disclosure being indicated by the followingclaims. All references, patents, and patent applications referred to inthis application are herein incorporated by reference in their entirety.

1. A method of treating a condition selected from the group consistingof pre-diabetes, diabetes, and metabolic syndrome in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of citrulline and a therapeuticallyeffective amount of leucine or a metabolite thereof.
 2. The method ofclaim 1, wherein the metabolite of leucine is hydroxyisocaproic acid(HICA) or beta-hydroxy-beta-methyl butyrate (HMB).
 3. The method ofclaim 1, wherein the therapeutically effective amount of citrulline andthe therapeutically effective amount of leucine or a metabolite thereofresult in a synergistic effect.
 4. The method of claim 1, wherein thecitrulline and the leucine or metabolite thereof are administered to thesubject using at least one regimen selected from the group consisting ofweekly, daily, two times a day, and three times a day.
 5. The method ofclaim 1, wherein the subject is administered a fixed amount of thecitrulline and the leucine or metabolite thereof.
 6. The method of claim1, wherein the subject is administered a weight-based dose of thecitrulline and the leucine or metabolite thereof.
 7. The method of claim6, wherein the weight-based dose is about 0.01 g/kg to about 0.3 g/kg ofthe citrulline and 0.01 g/kg to about 0.3 g/kg of the leucine ormetabolite thereof.
 8. The method of claim 6, wherein the weight-baseddose is 1.0 g/kg of the citrulline and 1.0 g/kg of the leucine.
 9. Themethod of claim 1, wherein a pharmaceutical composition is administeredto the subject that comprises both the therapeutically effective amountof the citrulline and the therapeutically effective amount of theleucine or metabolite thereof.
 10. The method of claim 1, wherein thetherapeutically effective amount of the citrulline and thetherapeutically effective amount of the leucine or metabolite thereofare administered orally.
 11. The method of claim 1, wherein the subjectis administered protein in conjunction with the citrulline and theleucine or metabolite thereof.